In normal skin hemidesmosomes tether keratinocytes to the basement membrane zone. The integrin alpha6beta4 resides at the core of each hemidesmosome and stabilizes cell interaction with laminin-5 in the extracellular matrix. However, there is also evidence that a6b4 integrin is involved in cell migration. In aim 1, we present preliminary data suggesting that specific keratinocyte migration behavior requires alpha6beta4 integrin which, through its ability to regulate the activity of the small GTPase Rac1 and the actin-severing protein cofilin, determines a specific organizational state of laminin-5 in the extracellular matrix. We propose to uncover precisely how beta4 integrin determines matrix organization, with a focus on dissecting signaling pathways leading to proper matrix assembly. In aim 2 we will identify those residues in the cytoplasmic tail of the alpha6 integrin subunit that determine the way cells assemble their matrix and which regulate cell motility. In addition, in aim 2 we will assess whether the tetraspanin molecule CD151 modulates the activity of alpha6beta4 integrin using knockout cells derived from mice engineered to lack expression of this membrane molecule. We will also test the hypothesis that the integrin-associated tetraspanin CD151 regulates the ability of laminin-5 receptors to organize their ligand in the matrix of keratinocytes and we will assess whether CD151 modulates the dynamic properties of alpha6beta4 integrin complexes along the substratum-attached surface of cultured keratinocytes. In aim 3 we propose to study epidermal cell migration on basement membranes assembled by wild type keratinocytes and keratinocytes lacking expression of alpha6 integrin, beta4 integrin or CD151 in vivo and in organotypic culture. The results from our three aims will provide mechanistic insight into how alpha6beta4 integrin heterodimers and their associated proteins contribute to tissue remodeling during wound healing and certain pathological conditions.